J. Embryol. exp. Morph. 83, 119-135 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
Patterns of cell division during visual streak
formation in the frog Limnodynastes dorsalis
ByL-A. COLEMAN, S. A. DUNLOP AND L. D. BEAZLEY
The Neurobiology Laboratory, Department of Psychology, University of
Western Australia, Nedlands, Western Australia, W.A. 6009
SUMMARY
The site and extent of cell division were determined in midlarval tadpoles, animals at
metamorphic climax and 2-month juvenile Limnodynastes dorsalis using untreated animals
and those injected with colchicine or pH]thymidine shortly before sacrifice. Mitosis was
restricted to the ciliary margin at all stages and there were significantly more dividing cells
nasally and temporally than dorsally and ventrally. In animals injected with [3H]thymidine and
killed at a subsequent stage, labelled cells were grouped at a distance from the ciliary margin
and were more frequent in nasal and temporal than in dorsal and ventral retina. These results
suggest that differing extents of mitosis around the ciliary margin, reflected in the number of
cells entering the ganglion cell layer, may largely underlie the postmetamorphic formation of
a visual streak observed using wholemounts.
INTRODUCTION
The distribution of cells in the retinal ganglion cell layer of Anura changes
after metamorphosis. In 1981 Dunlop & Beazley reported for Heleioporus eyrei
that the radial density gradient with high peripheral values characteristic of
tadpoles evolved to a horizontally aligned high-density visual streak in adults.
Similarly, Bousfield & Pessoa (1980) reported an accentuation of the area
centralis in postmetamorphic Hyla raniceps. Although less dramatic changes
were found in Xenopus laevis, densities in nasal and in particular temporal
peripheries were found to exceed other regions only after metamorphosis
(Dunlop & Beazley, 1984). In each species these changes in cell distribution were
accompanied by increases in both total cell number and retinal area.
Several hypotheses can be put forward to explain the events underlying visual
streak formation. Cell division might take place within the ganglion cell layer
although this is unlikely since at premetamorphic stages in amphibia, mitosis is
largely confined to the ciliary margin (Giucksmann, 1940; Hollyfield, 1968;
Straznicky & Gaze, 1971). As an exception Hollyfield (1971) observed some
mitosis in the inner nuclear layer of late larval and metamorphosing Xenopus.
However, if mitosis was limited to the ciliary margin, then the extent of cell
division could be greater nasally and temporally than elsewhere. Alternatively,
patterns of mitosis could be similar around the retina with cell death or migration,
120
L-A. COLEMAN, S. A. DUNLOP AND L. D. BEAZLEY
both known to occur widely in developing neural systems (Rakic, 1977; Lamb,
1977), playing roles in shaping the streak.
Recently Tay, Hiscock & Straznicky (1982) injected Xenopus with [3H]thymidine at metamorphic climax and examined retinae 2 months later. They
found a greater addition of cells to temporal retina than elsewhere, a result which
could underlie increased cell densities found in this region of wholemounts
(Dunlop & Beazley, 1984). However to begin to understand visual streak formation it is necessary to examine the retina of a species such as Limnodynastes
dorsalis which has more pronounced density gradients than Xenopus. In this
species we have analysed patterns of cell division to determine whether they
could explain changes in cell distribution. Our findings have been published in
abstract form (Coleman, Dunlop & Beazley, 1983).
METHODS
We examined sectioned retinae from tadpoles, animals at metamorphic climax
and juveniles in four conditions:- group (i) untreated, group (ii) treated with
colchicine 1-3 days before sacrifice, group (iii) injected with [3H]thymidine and
sacrificed within 1 day, group (iv) injected with [3H]thymidine as tadpoles or
animals at metamorphic climax and sacrificed after several weeks. Since mitosis
comprises only a small part of the cell cycle, colchicine was used to arrest cells
in metaphase allowing a better estimate of the size of the dividing cell population. [3H]thymidine, which is taken up during the S phase of cell division,
enabled us to identify cells dividing within a few hours of injection (Beach &
Jacobson, 1979). By examining the distribution of mitotic figures (groups i and
ii) and of labelled cells (group iii) the site of cell division could be established.
Furthermore the extents of cell division could be compared between nasal,
temporal, dorsal and ventral retina from counts of mitotic figures (groups i and
ii) and of labelled cells (groups iii and iv). Group (iv) was included because it was
the only series in which it was possible to consider solely those cells which finally
formed part of the ganglion cell layer.
We also prepared retinal wholemounts for tadpoles, animals at metamorphic
climax, juveniles and adults to document changes in the number and distribution
of cells in the ganglion cell layer and retinal area. In addition, axon totals were
estimated in a tadpole, juvenile and an adult.
Animals
Egg masses were collected from Kings Park, Perth in June/July and adults
from the metropolitan area. Tadpoles were fed Biorell fish food and kept at a
density of approximately 20/5 litres of spring water. At metamorphic climax
animals were transferred to shallow water and gravel. Postmetamorphic animals
were fed mealworms twice weekly ad libitum or by hand. A temperature of
22° ± 2°C and a 12 h light/dark cycle were maintained throughout.
Cell division during visual streak formation in frog
121
Tadpoles and animals at metamorphic climax were examined at stages
equivalent to 53-54 and 61-65 respectively in Xenopus (Nieuwkoop & Faber,
1956); juveniles were approximately 2 months postmetamorphosis and adults at
least 1-year old (Fig. 4). For injections, anaesthesia was by immersion in 0-1 %
MS222. Animals were sacrificed by decapitation while deeply anaesthetized with
MS222 (1 %) except for adults which received intraperitoneal injections of
Nembutal (0-7ml/gm body weight).
Studies of cell division
Injection procedures
Colchicine (10 or 100 mM) was administered intraperitoneally injecting 20/xl
for tadpoles and animals at metamorphic climax and 50jul for juveniles (group
ii). Tadpoles and juveniles were sacrificed after 24, 48 or 72 h whereas animals
at metamorphic climax proved sensitive and survival was limited to a maximum
of 15 h.
[3H]thymidine was injected intraperitoneally (10/iCi in 10/il, Amersham
specific activity 888GBq/mmol) and some animals at each stage were sacrificed
4 or 24 h later (group iii). Other injected tadpoles were killed either at metamorphic climax or as juveniles whilst some animals injected at metamorphic climax
were examined as juveniles (group iv).
Histology
Eyes were removed with surrounding tissue to assist orientation and fixed in
10 % buffered formalin (pH7-4) before being wax embedded, serially sectioned
and stained with haematoxylin and eosin. Right eyes were sectioned nasotemporally to reveal dorsal and ventral retina while dorsoventral sections of left eyes
allowed us to examine nasal and temporal retina. Slides for autoradiography
were dipped in NTB emulsion (Kodak), exposed for 10-14 days at 4°C and
developed in D19 before staining.
Analysis
To examine the site of cell division we noted the position of mitotic figures in
one out of every three sections of group (i) and in every section containing the
centrally placed optic nerve head (Fig. 5) of group (ii); the position of labelled
cells was observed in all sections of groups (iii) and (iv). To ensure that comparable sections were used for estimating numbers of dividing cells in nasal, temporal, dorsal and ventral retina, we analysed only those sections containing the
optic nerve head; equal numbers of sections for both eyes of each animal were
examined. In group (iv) sample grain counts were made and only the most
heavily labelled cells, considered to be generated within a few hours of injection
(Hollyfield, 1968; Meyer, 1978), were recorded. For statistical analysis, mean
numbers of mitotic figures or labelled cells were calculated for nasal, temporal,
dorsal and ventral retina. Results from individual animals were treated separately
122
L-A. COLEMAN, S. A. DUNLOP AND L. D. BEAZLEY
and an F test applied to determine if there were significant differences between
means. If so, differences were further analysed using a series of planned
orthogonal comparisons testing between poles of the same eye (i.e. nasal versus
temporal and dorsal versus ventral) as well as between eyes of the same animal
(i.e. nasal and temporal versus dorsal and ventral).
Retinal wholemounts and axon counts
Eyes were removed from normal animals and fixed for at least 1 week in 10 %
buffered formalin (pH7-4). Retinae were dissected and radial cuts made before
drying down, ganglion cell layer uppermost, on gelatinized slides and staining
with cresyl violet (Dunlop & Beazley, 1981). Retinae were oriented by the
ventral entry of the hyaloid artery. Pre- and post-staining areas were estimated
from tracings using a MOP-3 image analyser (Zeiss). Shrinkage ranged from
0-12 % and by reference to features such as the vascular tree was considered to
be confined largely to cut edges. The distribution of the total population in the
ganglion cell layer was determined by counting cells/(100 ^m) 2 sample area at
X1000 final magnification. Retinae were sampled systematically analysing
12-5 % of area for tadpoles and animals at metamorphic climax, 8 % and 5 % of
area for juveniles and adults respectively. Total cell number in the ganglion cell
layer was calculated by proportionality. To draw density profiles, cells/(100 jum)2
were also counted at intervals across the nasotemporal and dorsoventral axes
centering on the optic nerve head; in the adult the nasotemporal axis transected
the area centralis. Axons were counted from sample electron micrographs and
the total estimated by proportionality having estimated nerve area from a scanmode montage (Beazley & Dunlop, 1983).
RESULTS
Site of cell division
In untreated animals (group i), cells at all stages of mitosis were seen along the
sclerad edge of the ciliary margin although their frequency was low (Fig. l,i).
Fig. 1. (i-iii). Representative bright-field micrographs of temporal ciliary margins
in tadpoles, (i) In untreated animals (group i) mi to ticfigures(example circled) were
seen but there were seldom more than three per section, (ii) After colchicine treatment (group ii) many arrested metaphase figures (example circled) were seen along
the length of the ciliary margin which was extended compared to untreated animals,
(iii) After injection of pH]thymidine and sacrifice within 24 h (group iii) heavily
labelled cells (example circled) were seen throughout the depth of the ciliary margin.
Arrows indicate the ganglion cell layer, (iv) Equivalent dark- (A) and bright(B) field micrographs of labelled cells in nasal retina of a juvenile injected with
[3H]thymidine as a tadpole (group iv). In the ganglion cell layer heavily labelled cells
were seen, the most advanced of these (arrowed) being further from the optic nerve
head than were labelled cells in the inner and outer layers (arrowed). Positions of
labelled cells are indicated by arrows on a low-power micrograph of the section
(inset). Numbers of animals examined for groups i-iv were 14,14,8 and 9 respectively.
Haematoxylin & eosin (H & E). Scale bars i-iii: 20/zm, iv: 25[im, inset: 0-5 mm.
Cell division during visual streak formation in frog
•; *' *
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'
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123
124
L-A. COLEMAN, S. A. DUNLOP AND L. D. BEAZLEY
After exposure to colchicine (group ii) arrested metaphase figures accumulated
in this region; they had deeply stained clumped chromatin and a thin rim of clear
cytoplasm (Fig. l,ii). The ciliary margin was always longer after colchicine treatment (Fig. l,ii) although the numbers of arrested metaphase figures did not
correlate with dose or exposure time. Mitotic figures were not observed away
from the ciliary margin.
In all animals injected with [3H]thymidine and killed within 24 h (group iii),
label was almost exclusively limited to the ciliary margin and was found
throughout its depth (Fig. l,iii). Most labelled cells were spindle-shaped but a
few labelled mitotic figures were seen adjacent to the pigment epithelium. In
addition, small numbers of labelled cells were observed apparently randomly
distributed throughout the retina, their appearance and position suggesting they
were haematogenous.
Animals injected with [3H]thymidine and sacrificed at a subsequent stage
(group iv) presented a consistent picture in that all labelled cells occupied a
distinct band across the width of the nuclear layers at a distance from the ciliary
margin (Fig. l,iv; A & B). The most heavily labelled cells tended to be nearer
the optic nerve head. Labelled cells extended slightly more centrally in the inner
nuclear layer presumably reflecting the time at which cells left the mitotic cycle
(Morris, Wylie & Miles, 1976).
Numbers of dividing cells (Table 1)
All tadpoles injected with colchicine (group ii) had significantly (P<0-05)
more mitotic figures in nasal and temporal retina compared to dorsal and ventral.
In untreated tadpoles (group i) this difference was apparent but not significant.
Untreated and colchicine-treated animals at metamorphic climax and juveniles
had more mitoses in nasal and temporal compared to dorsal and ventral retina.
This difference was significant (P < 0-05) for all except one animal at metamorphic climax. At all stages lowest counts were usually in dorsal retina. In general
numbers of mitotic figures were highest at metamorphic climax and lowest in
juveniles. Our mitotic figure counts were reflected in the size of the ciliary margin
(Fig- 2).
Animals injected with [3H]thymidine and killed within 24 h (group iii) had
consistently more label in nasal and temporal than dorsal and ventral poles.
Density of label and packing of cells precluded counts of labelled cells in some
animals, particularly those at metamorphic climax. However two tadpoles and
a juvenile were suitable and in these there were significantly (P<0-05) more
labelled cells nasally and temporally than dorsally and ventrally. All animals
injected with [3H]thymidine and killed at a subsequent stage (group iv) showed
significantly (P < 0-05) more labelled cells in the ganglion cell layer nasotemporally than dorsoventrally. This result indicated that histogenetic events at the
ciliary margin analysed in groups (i-iii) were reflected in the number of cells
entering the ganglion cell layer (group iv).
Cell division during visual streak formation in frog
2A
/
i
*
• '
»2
&
Fig. 2. Ciliary margins of an untreated tadpole (A & B) and an untreated animal at
metamorphic climax (C & D). The ciliary margin was more extensive at metamorphic climax than in tadpoles, and at both stages the margin was longer nasally (B &
D) than dorsally (A & C). H & E. Scale bars 20//m.
EMB83
125
8-3
0-5
0-5
0-5
0-5
0-5
0-7
Juvenile
7-6
24-5
14-2
4-9
10-0
4-8
5-6
4-4
Tadpole
3-9
3-9
Colchicine-treated
(ii)
6-8
7-8
3-6
3-4
i
i
~r
i
i
i
~r
—
1
1-7
2-0
1-5
2-2
3-4
Metamorphic climax
14-5
28-6
16-8
6-9
36-6
9-5
25-3
15-6
6-8
14-0
1-7
1-8
1-0
0-7
1-8
1-0
1-5
1-5
1-0
1-1
1-4
0-9
2-8
1-2
— 3-4
1-1
V
0-8
N
Tadpole
V
Untreated
(i)
D
Stage Examined
Group
i
i
i
i
i
i
i
i
i
i
i
i
13-0
29-9
18-3
6-4
25-7
20t24
22*48
2 0 *48
1 4 *24
19f24
29
30
30
30
23
24
30
22
26
30
33
4-7
6-3
3-7
4-2
4-9
1-4
1-7
1-8
1-7
1-5
1-5
17
17
n
2-6
1-5
T
Table 1. Mean counts of mitotic figures (groups i&ii) and labelled cells(groupsHi c!£ iv) for each retinal axis
r
w
N
w
w
>
a
r
d
r
o
o
>
C/3
o
o
r
w
to
\
* • „ . • „ .
i
Juvenile
(injected at
metamorphic climax)
Metamorphic climax
(injected as tadpole)
Juvenile
(injected as tadpole)
Juvenile
Tadpole
u
25-5
u
1-8
2-1
3-7
4-3
2-0
2-6
i
i
i
i
i
4-4
3-7
3-3
5-8
8-9
2-3
5-0
2-7
2-6
4-6
1-8
1-9
12-8
12-4
33-1
11-1
7-7
4-3
3-3
5-3
7-9
7-1
6-9
6-4
T~
i
i
i
i
i
i
i
i
10
10
10
10
10
3-3
9-7
5-1
5-2
9-7
10
34-4
3-5
24§
11-2
23t
30t 72
48
22* 7 2
17*
17*
21-6
8-5
8-7
5-7
i
9-5
29-3
8-1
i
lit 5
28t9
iot 15
25-4
26*48
21* 5
34-5
24-7
23* 5
7-7
6-4
21-7
i
i
22-8
16-2
22-4
13-6
~
i
i
24-8
18-8
i
10-5 —
26-9 —
8-9
21-9
12-5
5-9
4-4
4-4
2-7
Juvenile
~
1
1
tlOO M l h' '
r post-injection intervals m hours are shown in superscript
t 4 h post-injection interval
§24h post-injection interval
D, V, N and T represent dorsal, ventral, nasal and temporal retina respectively. The bars indicate where differences between means were
significant (P<0-05), n refers to the number of sections examined for each pole.
* 1 0mM-colchicine
[3H]thymidine treated
(iv)
[ 3 H]thymidine treated
(iii)
6-5
23-1
190
13-5
18-2
Metamorphic climax
ts>
"S"
5-
1
S
3
5!'
g
128
L - A . COLEMAN, S. A. DUNLOP AND L . D . BEAZLEY
>V
Fig. 3. (A). The ganglion cell layer of a juvenile wholemount illustrating that most
cells were small and darkly stained. Only large cells (arrowed), which were in a
minority, had neuronal features such as clumped Nissl substance within pale
cytoplasm. Cresyl violet. Scale bar 10 jum. (B). Electron micrograph of an adult optic
nerve. The majority of axons were unmyelinated (open arrow) and fasciculated by
glial processes (*); a small myelinated axon is shown (solid arrow). Axon counts
suggested that throughout development many of the small cells seen in wholemounts
were indeed ganglion cells. Scale bar 0Retinal wholemounts and axon counts (Table 2)
The majority of cells in the ganglion cell layer had a similar appearance at
any stage with rounded dark nuclei and sparse cytoplasm (Fig. 3A). From
tadpole to juvenile, cell number and retinal area (Fig. 4) increased to similar
extents (x2-7 and x2-4 respectively) although between juvenile and adult, cell
addition (xl-8) was less than areal enlargement (x3-4). In tadpoles cell
densities were lowest centrally and dorsally. At metamorphosis and in juveniles
high-density patches extended from nasal and temporal peripheries toward the
Fig. 4. Graphs of total cell number in the ganglion cell layer ( • ) , retinal area after
staining (mm2, A) and optic axon number (O) for tadpoles (Td), animals at
metamorphic climax (M), juveniles (J) and adults (A). Mean cell number and retinal
area are plotted, the number of observations being shown next to the symbols;
standard deviations were small (Table 1). Animal lengths were: tadpoles (a) nose-totail 55 mm, metamorphic climax (b), juveniles (c) and adults (d) nose-to-anus
24-29 mm, 23-28 mm and 70 mm respectively. Tadpoles had the full complement of
labial teeth while these were absent at metamorphic climax although the mouth was
immature. Hindlimb buds had reached the foot paddle stage in tadpoles; at
metamorphic climax all 4 limbs were mature and the tail at various stages of resorption. Mean intervals between tadpoles and metamorphic climax and between this
stage and juveniles were 30 and 60 days respectively. Adults were considered to be
at least 1 year old since they were sexually mature (Main, 1957). Scale bars: (a-c)
0-5 cm; (d) lcm.
Cell division during visual streak formation in frog
129
optic nerve head while lower densities were observed dorsally and ventrally.
In adults a visual streak enclosed the area centralis in temporal retina; densities
were lower dorsally than ventrally. Changing cell distributions are shown as
isodensity contours and density profiles (Figs 5,6). Axon (Fig. 3B) counts were
Fig. 4
1L
2R
2L
499,000 ± 16,100
275,000 ±10,600
206,000 ± 8,800
101,000 ±9,800
X±S.D.
327,000
203,000
72,000
Axon No.
35
26
29
% Difference
cell & axon nos
63-7
59-2
62-8
15-1
15-3
20-4
19-6
20-8
7-9
7-0
5-8
8-7
8-2
13-3
12-9
13-1
16-2
16-1
Retinal
Area (mm2)
61-9 ±3-8
18-2 ±2-5
14-3 ±1-4
7-5 ±1-0
X±S.D.
Total cell and axon numbers were corrected to the nearest 1000, retinal areas to the nearest 0-1 mm2. Cell totals differed between sides by 9,
7,3 and 2% for tadpole 2, animal at metamorphic climax 1, juvenile 1 and adult 2 respectively. One percent offibreswere myelinated in the tadpole
and juvenile and 3% for the adult. Isodensity maps of tadpole 3R, animal at metamorphic climax 3R, juvenile 1R and adult 2L are shown in Fig. 5.
Adult
289,000
520,000
493,000
482,000
272,000
263,000
198,000
203,000
218,000
85,000
111,000
101,000
107,000
Tadpole
1L
2R
2L
3R
3L
Metamorphic 1R
climax
1L
2L
3R
3L
1R
Juvenile
1L
2L
3R
3L
No. of cells in
ganglion cell layer
Animal
Table 2. Cell numbers in the ganglion cell layer, optic axon counts and post-staining retinal areas
r
w
w
w
>
N
o
d
w
t-
o
o1
Cell division during visual streak formation in frog
131
6.5mrr
Fig. 5. Representative isodensity maps of cells in the ganglion cell layer for a tadpole
(a, 3 right eye), an animal at metamorphic climax (b, 3 right eye), a juvenile (c, 1
right eye) and an adult (d, 2 left eye). Numbers refer to cells per (100 /an)2 sample
area. The optic nerve head is indicated by a circle. V is ventral.
29 % (tadpole), 28 % (juvenile) and 35 % (adult) below their respective total cell
estimates.
DISCUSSION
Here we have examined the site and extent of cell division in the retina of
Limnodynastes dorsalis. This species resembled other Anura studied (Dunlop &
Beazley, 1981,1984) by developing from metamorphosis onwards a visual streak
within the ganglion cell layer.
Cell division was confined to the ciliary margin of Limnodynastes in agreement
with most other studies of both fish (Hollyfield, 1972; Meyer, 1978; Sharma &
Ungar, 1980) and amphibia (Straznicky & Gaze, 1971; Beach & Jacobson, 1979;
132
L-A. COLEMAN, S. A. DUNLOP AND L. D. BEAZLEY
220
200
ISO
160
140
Tadpole
MetamOrphic Climax
§
Juvenile
260
240
220
200
Adult
ONH
Distance from ONH (mm)
Fig. 6. Representative isodensity profiles from the wholemounts shown in Fig. 5. To
allow comparison with the adult the nasotemporal axis is laterally inverted for the
other animals. Numbers refer to cells per (100 jum)2 sample area along nasotemporal
(NT, • ) and dorsoventral (DV, • ) axes through the optic nerve head (ONH). Small
symbols represent values at biological edges.
Gaze, Keating, Ostberg & Chung, 1979; Tay et al. 1982). To our knowledge
there is only one report of mitosis away from the ciliary margin in amphibia, this
being in the inner nuclear layer of larval and metamorphosing Xenopus
(Hollyfield, 1971). In contrast mitosis has been reported in the outer nuclear
layer of several fish (Lyall, 1957; Ahlbert, 1976; Sandy & Blaxter, 1980; Johns
& Fernald, 1981; Johns, 1982). Asymmetric patterns of cell division around the
ciliary margin such as we report for Limnodynastes were found in late larval
Xenopus although in most studies only the dorsoventral axis was examined
(Straznicky & Gaze, 1971; Hollyfield, 1971; Jacobson, 1976; Straznicky & Tay,
1977; Beach & Jacobson, 1979; Gaze et al. 1979). We have demonstrated also
that patterns of mitosis at the margin are reflected in the number of cells entering
the ganglion layer in accord with the finding of asymmetric addition of cells to
this layer in metamorphosing Xenopus (Tay et al. 1982).
Variations in the extents of mitosis around the ciliary margin could be
Cell division during visual streak formation in frog
133
explained by differences in either the size of the precursor pool or cycle time. A
correlation between ciliary margin length and amounts of cell division argues for
the former. Such a conclusion would be in line with the finding of equal cycle
times reported for dorsal and ventral Xenopus retina (Beach & Jacobson, 1979).
It would seem probable that our finding of increased cell division at nasal and
temporal compared to dorsal and ventral poles (groups i-iii) directly accounts for
the greater addition of cells to the retinal ganglion cell layer nasally and temporally (group iv). These results apparently provide a sufficient explanation for the
changing distributions we have observed in the ganglion cell layer of
wholemounts. It is not necessary therefore to envisage major roles for other
cellular events such as death and migration in visual streak formation. Indeed
thorough examination did not reveal dying cells in our untreated or [3H]thymidine-injected material. We consider it unlikely that we have misidentified
dying cells, since they were readily distinguished in the group treated with
colchicine, known for its toxic effects (Hollyfield, 1968; Meller, 1981). Furthermore had there been a wave of cell death, cell numbers might have been expected
to drop at some stage, whereas we have reported a continual increase.
Migration of cells during visual streak formation is a more difficult issue to
resolve. Grafting of retinal segments between different marker mutants (Hunt
& Ide, 1977) might prove useful to examine migration from dorsal and ventral
retina into the streak or between nuclear layers. However we can exclude radial
migration since in animals injected with pHJthymidine and sacrificed after
several weeks' labelled cells were always closely grouped throughout the retinal
layers.
It is important to consider the part played by areal growth in changing cell
distributions since retinal area increased several fold from tadpole to adult.
Areal enlargement in the absence of asymmetric patterns of cell division could
have produced the high density patches seen in juveniles only if the dorso ventral
axis had grown more than the nasotemporal one. This did not occur. From our
wholemounts it is apparent that axes were of approximately equal length until
the juvenile stage. However between the juvenile and adult lesser extension of
the temporal compared to other axes may explain the development of an area
centralis in this region. Furthermore, the overall drop in cell density observed
between these stages is presumably a result of areal enlargement outstripping cell
addition.
This paper has considered developmental processes which might underlie
changing distributions of the total cell population in the retinal ganglion cell
layer. It would be of interest to extend these studies to analyse separately the
various cell types comprising this layer. The shortfall of axon to cell counts
suggests at least 30 % of cells are non-ganglion cells. In the frog Hyla moorei nonganglion cells represent a higher percentage of cells in dorsal and ventral
peripheries than elsewhere (Humphrey & Beazley, in press). If Limnodynastes
resembles Hyla in this respect, the implication is that cell types are added in
134
L-A. COLEMAN, S. A. DUNLOP AND L. D. BEAZLEY
different proportions around the ciliary margin. Thus we would predict a higher
percentage of ganglion cells would be generated at the nasal and temporal compared to dorsal and ventral poles. A combined [3H]thymidine and retrograde
horseradish peroxidase study would address this issue.
LDB is a Senior Research Fellow, National Health and Medical Research Council Australia
(NH&MRC). These experiments were performed in partial fulfilment of an honours degree
by L-A.C. This research was supported by NH&MRC grants 79/2087 and 82/0180 and the
Muscular Dystrophy Research Association of Western Australia. We are grateful for the use
of the Electron Microscopy Centre, University of Western Australia. We thank J. Durston,
J. Darby, M. Stevens and H. Jurkiewicz for histological, art and photographic assistance. C.
Pfaff, P. Basden and D. Anstey are thanked for typing the manuscript.
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{Accepted 27 April 1984)